Apoptotic protease activating factor 1 (Apaf-1)-independent cell death suppression by Bcl-2

Reportedly, antiapoptotic Bcl-2 family proteins suppress apoptosis by bindi ng to and inhibiting members of the CED-4 family of caspase activators. To explore this question, we used embryonic stem (ES) cells in which one (-/+) or both (-/-) copies of the gene encoding apoptotic protease activating fa ctor 1 (Apaf-1), a CED-4 homologue, were disrupted by homologous recombinat ion. Stable clones of heterozygous (-/+) and homozygous (-/-) Apaf-1 knocko ut ES cells that overexpressed Bcl-2 were generated. Withdrawal of serum gr owth factors or stimulation of heterozygous ES cells with staurosporine (ST S), ultraviolet (UV)B irradiation, etoposide (VP16), or cisplatin induced a poptosis followed by cell death (determined by failure to exclude propidium iodide dye). These cell death stimuli also induced activation of several t ypes of caspases and loss of mitochondrial membrane potential (Delta Psi) i n heterozygous (+/-) Apaf-1 knockout ES cells. In addition, overexpression of Bcl-2 protected against these events in Apaf-1-expressing ES cells. In c ontrast, STS, UVB, and VP16 induced little or no caspase activation and apo ptosis in homozygous (-/-) Apaf-1 knockout ES cells. Nevertheless, Apaf-1-d eficient ES cells subjected to these cell death stimuli or deprived of grow th factors did eventually die through a nonapoptotic mechanism associated w ith loss of Delta Psi. Moreover, Bcl-2 overprotection preserved Delta Psi, reduced the percentage of Apaf-1(-/-) ES cells undergoing cell death, and i ncreased clonigenic survival. The extent of Bcl-2-mediated cytoprotection w as not significantly different for heterozygous (-/+) versus homozygous (-/ -) Apaf-1 knockout cells. Furthermore, although Bcl-2 could be readily coim munoprecipitated with Bax, associations with Apaf-1 were undetectable under conditions where Apaf-1 interactions with procaspase-9 were observed. We c onclude that Bcl-2 has cytoprotective functions independent of Apaf-1, pres erving mitochondrial function through a caspase-independent mechanism.